β-peptide conjugates: Syntheses and CD and NMR investigations of β/α-chimeric peptides, of a DPA-β-decapeptide, and of a PEGylated β-heptapeptide

Article abstract of DOI:10.1002/hlca.200900325

β³-Peptides consisting of six, seven, and ten homologated proteinogenic amino acid residues have been attached to an α-heptapeptide (all d-amino acid residues; 4), to a hexaethylene glycol chain (PEGylation; 5c), and to dipicolinic acid (DPA de

Full text of DOI:10.1002/hlca.200900325

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b-Peptide Conjugates: Syntheses and CD and NMR Investigations of
b/a-Chimeric Peptides, of a DPA-b-Decapeptide, and of a PEGylated
b-Heptapeptide
by James Gardiner1), Raveendra I. Mathad2), Berhard Jaun, Jꢀrg V. Schreiber3), Oliver Flçgel4), and
Dieter Seebach*
Laboratorium fꢀr Organische Chemie, Departement Chemie und Angewandte Biowissenschaften, ETH
Zꢀrich, HCI Hçnggerberg, Wolfgang-Pauli-Strasse 10, CH-8093 Zꢀrich
(phone: þ 41-44-632-2990; fax: þ 41-44-632-1144; e-mail: seebach@org.chem.ethz.ch)
b3-Peptides consisting of six, seven, and ten homologated proteinogenic amino acid residues have
been attached to an a-heptapeptide (all d-amino acid residues; 4), to a hexaethylene glycol chain
(PEGylation; 5c), and to dipicolinic acid (DPA derivative 6), respectively. The conjugation of the b-
peptides with the second component was carried out through the N-termini in all three cases. According
to NMR analysis (CD3OH solutions), the (M)-314-helical structure of the b-peptidic segments was
unscathed in all three chimeric compounds (Figs. 2, 4, and 5). The a-peptidic section of the a/b-peptide
was unstructured, and so was the oligoethylene glycol chain in the PEGylated compound. Thus, neither
does the appendage influence the b-peptidic secondary structure, nor does the latter cause any order in
the attached oligomers to be observed by this method of analysis. A similar conclusion may be drawn
from CD spectra (Figs. 1, 3, and 5). These results bode well for the development of delivery systems
involving b-peptides.
1. Introduction. – Many natural products are conjugates made up of a mixture of
peptide segments, polyketides, sugars, or fatty acids. While these various structures may
not all play a role in promoting a key biological conformation or be part of an essential
pharmacophore, they are nevertheless important for the ability to impart stability,
solubility, or the correct locational recognition motif to promote optimal biological
function. Several pharmaceutical agents also make use of additional conjugation to
impart favorable physical properties on an effective but normally short-lived molecule
(cf. polyethylene glycol (PEG) chains to enhance half-life and increase solubility).
In an effort to explore the possibility of using b-peptides as delivery systems for
drugs, we aimed to prepare b-peptidic conjugates of b-peptides (consisting of
homologated proteinogenic amino acid residues) that would normally adopt a 314-
helix conformation in solution [1 – 3] to investigate whether the nonpeptidic segment
1
)
Postdoctoral Research Fellow at ETH-Zꢀrich (2004 – 2007), financed by the New Zealand
Foundation for Research Science and Technology (No. SWSS0401).
Part of the Ph.D. Thesis of R. I. M., ETH Dissertation No. 17209 (2007), financed by Swiss National
Science Foundation (No. 200020-109065).
Part of the Ph.D. Thesis of J. S., ETH Dissertation No. 14298 (2001).
Postdoctoral Research Fellow at ETH-Zꢀrich (2004 – 2006), financed by the Deutsche Forschungs-
gemeinschaft, by the Swiss National Science Foundation (No. 200020-100182), and by Novartis
Pharma AG, Basel.
2
)
3
4
)
)
ꢁ 2009 Verlag Helvetica Chimica Acta AG, Zꢀrich

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would have an effect on the stability of the helix. The CD- and NMR-solution structure
analyses of the b-peptides, with and without the conjugate, would be used to investigate
if the stability of the helix was influenced in any way, and whether or not the b-peptidic
helix would induce secondary structures in an attached a-peptide or in an oligo-
ethylene glycol moiety.
2. a/b-Chimeric Peptides. – Peptides with ꢂheterogeneousꢃ backbones (ꢂmixedꢃ
peptides) consisting of a-, b-, g-, and/or d-amino acid residues have recently moved into
the focus of interest [4]. We had wondered for a long time3) whether an (M)-314-helix-
forming b-peptide would influence the folding of an attached a-peptide, i.e., whether
the increased helical propensity of the b-peptide portion could somehow promote, or
ꢂforceꢃ, helicity in the a-peptidic segment of such a chimeric peptide. Since the a-
peptide composed of l-amino acid residues folds to a right-handed helix, while the b-
peptide consisting of l-b-amino acids folds to a left-handed helix, we decided to
perform the test with an a-peptide built of d-amino acid moieties, so that (M)-helicity
could possibly be attained along the whole length of the ꢂmixedꢃ-peptide chain5)6).
For structural investigations of such chimeric b/a-peptides that may adopt a 314/
3.613-helical motif, an appropriate sequence with intrinsic folding propensity was
needed. We have previously shown that b-peptides composed of as few as six l-b-amino
acid residues with the side chains of Val, Ala, and Leu fold into a (M)-314-helical
structure [1][2], and Karle et al. had reported that a-peptides containing a-amino-
isobutyric acid (Aib) residues adopt a helical structure (3.613- and/or 310-helix) in the
solid state [7]. Thus, tridecapeptide 3 was prepared by fragment coupling in solution of
the Aib-containing a-peptide 1c with the b-peptide 2 (see Exper. Part). The b-peptidic
ꢂfragmentꢃ 2 exhibits a typical CD pattern of a (M)-314-helix [1] (a trough at 215 nm, not
shown here), and the a-peptidic ꢂfragmentꢃ 1 consisting of all d-a-amino acids with a
central Aib residue gives rise to positive-only CD spectra (Fig. 1,a) of various
intensities, depending upon its state of protection7). The ꢂmixedꢃ a,b-peptide 3, on the
other hand, shows the expected trough at 215 nm, albeit weak, as compared to a
mixture of its precursors, the a-peptide 1c and the b-peptide 2 (Fig. 1,b); a summation
of the CD curves of 1c and 2 results in a very similar picture. With due caution8), we
may conclude from the CD spectra that there is no influence of the b-peptidic on the a-
peptidic secondary structure, neither in a mixture (intermolecular interactions might
have shown up) nor in the chimeric a/b-peptide 3 (intramolecular interactions).
5
)
Natural peptides can also fold to a left-handed helix, of which short 2- to 3-pitch-long segments are
seen in many proteins (see the Ramachandran diagram in textbooks of peptide and protein
chemistry).
Furthermore, there is a right-handed 310-helix of a-peptide chains. This helix is the preferred folding
pattern of peptides consisting of geminally disubstituted a-amino-acid residues, such as Aib
(aminoisobutyric acid) or Iva (isovaline or 2-amino-2-methylbutanoic acid [5]). For a comprehen-
sive treatment, see the recent monograph ꢂPeptaibiotics – Fungal Peptides Containing a-Dialkyl a-
Amino Acidsꢃ [6].
6
)
7
8
)
A (P)-3.613-helical structure was observed for the fully-protected a-heptapeptide Boc-l-Val-l-Ala-
l-Leu-Aib-l-Val-l-Ala-l-Leu-OMe in crystals; however, NMR evidence suggests conformational
variability in solution [7].
)
In our experience [2b][8], CD-only analysis of b-peptides can be misleading.

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An NMR-solution structural analysis of the a/b-peptide 3 was not possible, due to
the fact that it contains, besides Aib, amino acid residues with only three different types
of side chains (Me, i-Pr, i-Bu). We, therefore, prepared (by solid-phase synthesis on
Rink amide AM resin; see Exper. Part) the tridecapeptide 4 with the following features:
i) there are no two identical residues in the a- and b-peptidic segment; ii) there are five
functionalized side chains for good solubility in a protic solvent; iii) the b-peptidic part
carries a Glu and a Lys side chain in (i) and (i þ 3) positions for salt-bridge stabilization
of a 314-helix [2b][9]; iv) there are two amino acids (Phe and Trp) with aromatic groups
in the side chain for better HPLC detection; v) the indolyl group of Trp provides
fluorescence; and vi) there is a helix-inducing Aib residue in the center of the a-
heptapeptidic d-amino acid sequence. A detailed NMR analysis of the tridecapeptide 4
was carried out, including a structure calculation by simulated annealing using the
XPLOR-NIH protocol, and distance as well as dihedral angle constraints as derived
1
from the NOE data and coupling constants (for complete assignments of the H- and
13C-NMR signals, see Tables 1 and 2 in the Exper. Part). The ten structures lowest in
calculated energy, and without NOE or dihedral angle constraint violations are
displayed in Fig. 2. This structural bundle reveals that all six b3homo-amino acid
residues from the N-terminus to b3hIle6 are arranged in two complete turns of a (M)-
314-helix, whereas the NMR data for the seven a-amino acid residues do not constrain
this C-terminal part to any of the secondary structures known for a-peptides. This is
3
corroborated by the J(HN,Ha) values for residues Phe7 to Ala-NH213, which range
from 5.9 to 7.8 Hz and indicate a random coil arrangement of the backbone in the a-
peptidic part. Therefore, the answer to the question of whether a b-peptide helix can
induce helicity in an a-peptide is, in this case, no. This result does, however, provide a
unique demonstration of the difference between the folding propensities of a- and b-
peptides: even with a helix-inducing Aib residue the a-peptidic portion is disordered,
while the b-peptidic portion shows an ordered 314-helix. How could one better
demonstrate this difference than by putting an a- and b-peptide together covalently in
the same molecule and examining their conformations ꢂunder identical conditionsꢃ.
3. PEGylated b-Peptide. – Attachment of poly- or oligoethylene glycol chains
(PEGylation)9) to therapeutic proteins, peptides (cf. A), and small-molecule
therapeutics is a well-established method used to influence solubility, stability,
immunogenicity, pharmacokinetics, and mode of action [10]10). Unlike in polyethy-
9
)
Strictly speaking, polyethylene glycol, prepared by polymerization of ethylene oxide (n C2H4O þ
HX ! H-(OCH2CH2)n-X) is not a uniform compound, but a mixture of oligoethylene glycols with a
Gaussian distribution of molecular weights. Our peptide derivative 5c carries a hexaethylene glycol
chain, attached to the N-terminus through a b-hydroxypropanoyl group. Still, following common
practice, herein we use the term ꢂPEGylated b-heptapeptideꢃ.
One example is PEGylated interferon alfa-2a (40 kD; commercial name PEGASYS), an antiviral
drug developed by F. Hoffmann-La Roche; it has a dual mode of action – both against viruses and
on the immune system. This drug is approved around the world for the treatment of chronic
hepatitis C (including patients with HIV co-infection, cirrhosis, ꢂnormalꢃ levels of ALT) and has
recently been approved (in the EU, USA, China, and many other countries) for the treatment of
chronic hepatitis B (see http://www.pegasys.com/about-pegasys/how-pegasys-works.aspx). For a
review article on PEGylated proteins, peptides and non-peptidic drugs, see [10].
10
)

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lene, the chain in PEG is not arranged in a zig-zag conformation B but is helical, due to
the gauche effect [11a], which generally favors synclinal conformations of ethane
moieties carrying two heteroatoms (X-CꢀC-Y, see C). Of course, the two enantio-
meric conformations (þ)-sc and (ꢀ)-sc C in a PEG chain [11b – d] lead to helices of
opposite (P)- or (M)-helicity, and, in a random PEG sample, there will be equal
amounts of the enantiomeric helical conformations in rapid equilibrium at room
temperature (low barrier to rotation around the ethane CꢀC bonds)11). We wondered
whether there would be interaction between a b-peptidic 314-helix and a PEG unit in
the same molecule, which could lead to an induced helical arrangement of the ethylene
glycol chain, and which also could lead to a stabilization of the peptide helix.
The b-heptapeptide 5a was chosen to look for such an interaction; it was prepared
via solid-phase peptide synthesis using standard Fmoc strategy (see Exper. Part); a
PEG chain was attached to its N-terminus (! 5c) under peptide-coupling conditions on
the resin (HATU/Hꢀnig base), using Fmoc-NH(CH2CH2O)6(CH2)2CO2H12); for
comparison, the N-Ac derivative 5b was also prepared.
CD Analysis (Fig. 3) of 5a, 5b, and 5c in MeOH (0.2 mm) indicates that all three
peptides have helical structures, with characteristic negative Cotton effects near
215 nm. The intensity of the Cotton effect increases in the order 5a (free N-terminal
amino group) < 5b (acetylated N-terminus) < 5c (PEG-peptide). This is the more
remarkable, since the ꢂmolecular b-peptidic contentꢃ decreases from 5a (MW 849.5) to
5b (MW 913.5) to 5c (MW 1184.7), and this might8) be interpreted as a helix-stabilizing
effect of the PEG appendage.
A detailed NMR analysis of the N-Ac derivative 5b and of the PEGylated b-peptide
5c was then performed (see Tables 3 – 6 in the Exper. Part) to see whether interactions
could be detected between the peptide and the PEG part of the molecule on the NMR
time scale. The NMR structure of the N-acetylated b-heptapeptide 5b (Fig. 4,a) in
MeOH has the expected 314-helical folding observed for similar b3-peptides [1 – 3]. The
PEGylated compound 5c (Fig. 4,b) adopts a helical conformation of the b-peptidic
portion, almost identical to that of the N-Ac derivative 5b, which does not appear to
interact at all with the covalently attached PEG chain (i.e., no NOEs were observed
between NMR signals associated with the PEG portion of the molecule and those
originating from the peptide portion). It must be emphasized that the globular-type
11
)
In a recent communication the synthesis of discrete ethylene glycol oligomers (of up to 48
CH2CH2O units) was described; a hexadecamer monomethyl ether gave single crystals consisting of
right- and left-handed 310-helices (X-ray structure analysis) [12a], confirming the structure found by
polymer chemists (stretched-fiber X-ray diffraction) a long time ago [12b – d].
Commercially available from Quanta Biodesign Ltd.
12
)

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Fig. 3. CD Spectra of b-heptapeptide derivatives 5a – 5c. The spectra were recorded in MeOH (0.2 mm)
and are not normalized.

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arrangement of the PEG chain with no defined conformation as shown in Fig. 4,b, is an
artifact of the simulated annealing calculation: since the H-atoms of the OCH2CH2O
1
moieties of the PEG chain are almost isochronous in the H-NMR spectrum, no
dihedral angle or NOE constraints within the PEG chain could be derived
experimentally, which leads to a random distribution of all local staggered conforma-
tions in the calculations.
Fig. 4. NMR-Solution structures of a) the acetylated b-heptapeptide 5b, and b) the PEGylated b-
heptapeptide 5c, in CD3OH. The structure of 5b is shown in top view along the helix axis and in side view
perpendicular to this axis. The structure of 5c is shown perpendicular to the helix axis.
Thus, the NMR analysis was not able to confirm an interaction between the b-
peptidic and the ethylene glycol parts in compound 5c that was suggested by the CD
analysis. The complete absence of NOEs between the PEG signals and the b-peptide
signals indicates that the PEG chain does not prolongate the helical turn at the N-
terminus of the peptide. However, this does not preclude that, within the PEG chain, a

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helical secondary structure such as seen recently in PEG oligomers by X-ray-analysis
(see Footnote 11 above) might be present.
4. The Dipicolinic Acid (DPA) Derivative 6. – Lastly, to explore the potential of
small-molecule b-peptide conjugates, we chose to attach pyridine-2,6-dicarboxylic acid
(¼dipicolinic acid; DPA) to a b-decapeptide designed to adopt a 314-helix in MeOH
(compound 6 in Fig. 5). The high affinity of DPA for Tb3þ, resulting in intense
luminescence under UV excitation, forms the basis for a number of biologically
relevant assays including cell permeability [13] and bacterial spore detection (cf.
Anthrax [14]). Such DPA-peptide derivatives have previously been used by us to
investigate the permeation of a- and b-oligopeptides through lipid bilayers [15] and
also as potential somatostatin mimics [4b]. The b-peptidic segment of the peptide
conjugate 6 was designed to include a potential salt bridge between the side chain in
positions 5 (bhGlu) and 8 (bhLys) of the sequence to assist in stabilizing helical
secondary structure. Compound 6 was prepared via solid-phase peptide synthesis using
Fmoc strategy. N-Terminal attachment of DPAwas carried out under standard coupling
conditions (HATU/DIPEA) using pyridine-2,6-dicarboxylic acid mono-benzyl ester
[15]. Cleavage of the peptide from the resin, followed by hydrogenolysis of the benzyl
group and purification by RP-HPLC, gave the peptide 6 of > 98% purity.
The CD spectrum of peptide derivative 6 in MeOH (0.2 mm) shows an intense
negative Cotton effect near 215 nm indicative of a helical conformation (Fig. 5,b). The
NMR solution-structure analysis confirmed that compound 6 adopts the expected 314-
helical conformation in CD3OH (Fig. 5,c and d, and Tables 7 and 8 in the Exper. Part).
The fact that NOEs were observed between the H-atoms of the Leu side chain of
residue 3 and HꢀC(3) of the N-terminal pyridine ring (Fig. 5,a) suggests some
additional degree of order at the N-terminus including the pyridine-2,6-dicarboxylic
acid moiety.
It is, therefore, clear that covalent attachment of DPA to medium-length b-peptides
does not lead to destabilization of the 314-helix in MeOH (good news for the
development of small-molecule-b-peptide conjugates). In turn, for DPA-conjugates
such as 6, the b-peptidic sequence does not interact with the terminal aromatic
carboxylic acid group in any way, confirming that, in solution, the DPA moiety adopts a
conformation, which may be favorable for use in its associated biological assays.
5. Conclusions. – We have shown that the attachment of conjugates (DPA, PEG, a-
peptides) does not appear to affect the helical conformation of b-peptides designed to
adopt a 314-helix in MeOH. This has been demonstrated for the DPA-conjugated b-
peptide 6 and for the PEGylated conjugate 5c. These studies bode well for the future
attachment of drug-like molecules (such as antibiotics, DNA and RNA13), etc.) to b-
peptides for development as delivery systems and potential therapeutics.
We have also shown that covalent attachment of an a-peptidic sequence, via an
amide bond, to a b-peptidic sequence (to give a chimeric b/a-peptide) does not affect
the nature or stability of the 314-b-peptide helical segment. While we had originally
designed the chimeric b/a-sequence to induce helicity in the a-peptide segment, using
13
)
See chapter 10.5 in [2b] and Chapter 3 in [2d], and refs. cit. therein.

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the b-peptide segment, the NMR solution-structure analysis showed that this did not
occur. However, this result clearly, and quite dramatically, displays the greater inherent
stability of a b-peptide helix over that of an a-peptidic helix, in what could be called an
intramolecular comparison.
We thank Albert K. Beck for his invaluable help in preparing the manuscript of this work.
Experimental Part
Abbreviations. Boc: (tert-butoxy)carbonyl, CD: circular dichroism, DIPEA: N-ethyl-N,N-diisopro-
pylamine (EtN(i-Pr)2), DMAP: 4-(dimethylamino)pyridine, DPA: pyridine-2,6-dicarboxylic acid, EDC:
1-ethyl-3-[(dimethylamino)propyl]carbodiimide, Fmoc: [(9H-fluoren-9-yl)methoxy]carbonyl, HATU:
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HOBt: 1-hydroxybenzo-
triazole, h.v.: high vacuum (0.01 – 0.1 Torr), MALDI: matrix-assisted laser desorption ionisation, MeIm:
1-methyl-1H-imidazole, NMM: N-methylmorpholine, SPPS: solid-phase peptide synthesis, TFA:
trifluoroacetic acid, TIS: triisopropylsilane ((i-Pr)3SiH), TNBS: 2,4,6-trinitrobenzenesulfonic acid.
General. Side-chain-protected Fmoc-a- and Fmoc-b3-amino acids were purchased from Fluka. Wang
resin and Rink Amide AM resin were purchased from Novabiochem. NMR: Chemical shifts d are given
in ppm relative to resonances of solvent (1H: 3.31 ppm for CD3OD; 13C: 49.15 ppm for CD3OD),
coupling constants J are given in Hz. MS: VG Tribrid (EI), Bruker Reflex (MALDI), or IonSpec Ultima
4.7 TFT Ion Cyclotron Resonance (ICR, MALDI-HR-MS, in a 2,5-dihydroxybenzoic acid matrix) mass
spectrometer; in m/z (% of basis peak). Anal. HPLC: Merck HPLC system (LaChrom, pump type L-
7150, UV detector L-7400, interface D-7000, HPLC manager D-7000). Prep. HPLC: Merck/Hitachi
HPLC system (pump type L-6250, UV detector L-4000) or Merck HPLC system (LaChrom, pump type
L-7150, UV detector L-7400, interface D-7000, HPLC00 manager D-7000). TFA for anal. and prep. RP-
HPLC was UV-grade quality (> 99% GC). Lyophilization: Hetosicc cooling condenser with h.v. pump to
obtain the peptides as their TFA salts.
Loading of Wang Resin: General Procedure 1 (GP 1). To a soln. of the Fmoc-protected b-amino acid
(3 equiv.), in dry CH2Cl2 (5 ml) under Ar, was added Melm (2.25 equiv.) and MSNT (3 equiv.) The
mixture was stirred for 2 min and then added to the pre-swollen resin (CH2Cl2 for 1 h), and the
suspension was mixed by Ar bubbling for 4 h. The resin was then filtered, washed with CH2Cl2 (4 ml, 6 ꢁ
1 min), and dried under h.v. overnight. The loading was determined by measuring the absorbance of the
benzofulvene piperidine adduct: two aliquots of the Fmoc-amino acid resin were weighed exactly
(m1(resin) and m2(resin) [mg]) and suspended in an exact amount of piperidine solution (20% in DMF)
in volumetric flasks (V1 ¼ V2 ¼ 10 [ml]). After 30 min, the mixtures were transferred to a UV cell, and the
absorbance (A) was measured relative to a blank piperidine soln. (20% in DMF) at 290 nm. The
concentrations (c1 and c2, [mm]) of the benzofulvene – piperidine adduct in soln. were determined using a
calibration curve [16]. The loading (Subst.) was then calculated according to Eqn. 1:
Substn [mmol/g resin] ¼ cn · Vn/{mn(resin) ꢀ [cn · Vn · (MWꢀ 18)/1000]}
(MW¼ molecular weight of the Fmoc-protected amino acid).
(1)
The yield for the resin attachment (loading yield) was determined by Eqn. 2:
Loading yield ¼ [(Subst1 þ Subst2)/2]/Substtheor.
(2)
Anchoring of N-Fmoc-Protected Amino Acids on a Rink Amide Resin: General Procedure 2 (GP 2).
The resin was placed into a dried manual SPS reactor, swelled in DMF (20 ml/g resin) for 1 h, and washed
with DMF (6 ꢁ 5 ml). The resin was deprotected according to GP 4 and coupled with the first amino acid
according to GP 5.
Capping of Free Amino Groups: General Procedure 3 (GP 3). The peptide – resin was covered with
DMF (20 ml/g resin), and the unreacted amino groups were capped using Ac2O (10 equiv.) and DMAP

Helvetica Chimica Acta – Vol. 92 (2009)
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(0.1 equiv.) dissolved in DMF (0.1 ml/mmol Ac2O) for 1 – 2 h under Ar bubbling. The resin was then
washed with DMF (20 ml/g resin, 5 ꢁ 1 min) and with CH2Cl2 (20 ml/g resin, 5 ꢁ 1 min).
Fmoc Deprotection: General Procedure 4 (GP 4). The Fmoc deprotection was carried out using 20%
piperidine in DMF (4 ml, 4 ꢁ 10 min) under N2 bubbling. After filtration, the resin was washed with DMF
(5 ml, 4 ꢁ 1 min) and CH2Cl2 (5 ml, 4 ꢁ 1 min).
Coupling of Amino Acids on Wang, Rink Amide/Amide AM Resin: General Procedure 5 (GP 5).
Fmoc Deprotection was carried out according to GP 4. Solid-phase synthesis was continued by sequential
incorporation of Fmoc-protected amino acids. For each coupling step, the resin was treated with a soln. of
Fmoc-protected amino acid (3 equiv.), HATU (2.9 equiv.), and DIPEA (6 equiv.) in DMF (5 ml) for 1 –
2 h. Monitoring of the coupling reaction was performed with the TNBS test [17]. In case of a positive
TNBS test (indicating incomplete coupling), the suspension was allowed to react for a further 1 – 2 h, or
retreated with the same Fmoc-protected b-amino acid (2 equiv.) and coupling reagents. After complete
coupling, the resin was washed with DMF (5 ml, 5 ꢁ 1 min). The cycle was then repeated until all
remaining amino acids were incorporated.
Side-Chain Deprotection and Cleavage of Peptides from Wang/Rink Amide/Rink Amide AM Resin:
General Procedure 6 (GP 6). The dry peptide/peptide – resin was treated with a soln. of TFA/TIS/H2O
95 :2.5 :2.5 (10 ml) for 3 h. The resin was filtered, washed with TFA (2 ꢁ 5 ml), and the org. phase was
concentrated under reduced pressure. The crude peptide, which formed upon addition of cold Et2O to the
oily residue, was collected, dried under h.v., and stored at ꢀ 208 before being purified by RP-HPLC.
HPLC Analysis and Purification of Peptides: General Procedure 7 (GP 7). RP-HPLC Analysis was
performed with a Macherey-Nagel C18 column (Nucleosil 100-5 C18 (250 ꢁ 4 mm)) or a Macherey-Nagel
C8 column (Nucleosil 100-5 C8 (250 ꢁ 4 mm)) using a linear gradient of A (MeCN) and B (0.1% TFA in
H2O) at a flow rate of 1 ml/min with UV detection at 220 nm. Retention time (tR) in min. Aliquots of the
crude products were purified by prep. RP-HPLC with a Macherey-Nagel C18 column (Nucleosil 100-7 C18
(250 ꢁ 21 mm)) or a Macherey-Nagel C8 column (Nucleosil 100-7 C8 (250 ꢁ 21 mm)), using a gradient of
A and B at a flow rate of 10 ml/min with UV detection at 220 nm, and then lyophilized to give the pure
peptide as a TFA salt (purity > 95%).
Benzyl Deprotection: General Procedure 8 (GP 8). The Bn-protected peptide was dissolved in
MeOH (5 ml) under N2, and Pd/C (10% (w/w)) was added. The apparatus was evacuated, flushed with
H2 (3ꢁ), and the soln. was stirred vigorously under H2 (balloon) for 12 h. The soln. was filtered through
Celite, which was washed twice with MeOH, and the combined solns. were concentrated under reduced
pressure to give the crude product.
Boc Deprotection: General Procedure 9 (GP 9). Similarly to the reported procedure [18], the Boc-
protected compound was dissolved in CH2Cl2 or CHCl3 (0.06 – 0.55m) and cooled to 08 (ice-bath). An
equal volume of TFA was added, and the mixture was allowed to warm to r.t. and then stirred for further
1.5 – 3 h. Concentration under reduced pressure and drying of the residue under h.v. yielded the crude
TFA salt, which was, if not otherwise mentioned, used without further purification.
Peptide Coupling with EDC: General Procedure 10 (GP 10). GP 10a. A stirred soln. of the TFA salt
(1 equiv.) in THF, CH2Cl2, or CHCl3 (0.1 – 0.5m) at 08 (ice bath) was treated with the Boc-protected
fragment (1.0 – 2.5 equiv., added as solid or as a soln. in CHCl3 (0.5m)), NMM (2.7 – 3.5 equiv.), HOBt
(1.1 – 2.5 equiv.), and EDC (0.9 – 2.5 equiv.). The soln. was either stirred for 1 – 4 h at 08 (ice-bath) then at
r.t. for 26 h – 4.5 d, or was directly allowed to warm to r.t., and stirring was continued for 15 h – 3 d. The
mixture was diluted with CH2Cl2 and washed with 1n HCl, aq. sat. NaHCO3, and NaCl soln. The org.
phase was dried (MgSO4) and evaporated, and the residue was, if not otherwise mentioned, purified by
FC and/or recrystallization.
GP 10b. According to [19], a stirred soln. of the appropriate amino compound, TFA, TsOH, or HCl
salt (1.0 equiv.) in CHCl3 (0.1 – 0.5m) at 08 (ice bath) was treated successively with Et3N (1.3 – 5.0 equiv.),
HOBt (1.0 – 3.1 equiv.), the Boc-protected fragment (1.0 – 2.6 equiv., added as solid or as a soln. in CHCl3
(0.5 – 0.6m)), and EDC (1.2 – 3.1 equiv.). The mixture was allowed to warm to r.t., and stirring was
continued for 16 h – 4 d. Subsequent dilution with CHCl3 was followed by thorough washing with 1n HCl,
aq. sat. NaHCO3, and NaCl soln. The org. phase was dried (MgSO4) and then concentrated in vacuo. FC
and/or recrystallization yielded the pure peptide.

2710
Helvetica Chimica Acta – Vol. 92 (2009)
Preparation of 1a – 1d. Boc-d-Ala-d-Leu-OBn. According to [20], a soln. of Boc-d-Ala-OH (3.73 g,
19.7 mmol) in THF (40 ml) at ꢀ 158 was treated with NMM (2.20 ml, 20.0 mmol) and isobutyl
chloroformate (2.60 ml, 19.9 mmol). After 15 min, a cold (08 bath temp.) soln. of TsOH · H-d-Leu-OBn
(7.28 g, 20.1 mmol) and NMM (2.20 ml, 20.0 mmol) in THF (40 ml) was added dropwise. After 1 h at 08,
the ice-bath was removed, and the soln. was allowed to warm to r.t. Stirring was continued for 23 h. The
mixture was then diluted with AcOEt and washed with 5% citric acid, sat. aq. NaHCO3, and sat. aq. NaCl
soln. All aq. layers were additionally extracted twice with AcOEt. The combined org. layers were dried
(MgSO4) and evaporated to yield Boc-d-Ala-d-Leu-OBn (7.45 g, 96%), which was used without further
purification. 1H-NMR Data in agreement with those in [20].
Boc-d-Val-d-Ala-d-Leu-OBn. Boc-d-Ala-d-Leu-OBn (7.30 g, 18.6 mmol) was deprotected in
CH2Cl2 (37 ml) according to GP 9 for 3 h. To a soln. of Boc-d-Val-OH (4.03 g, 18.5 mmol) in THF
(27 ml), NMM (5.75 ml, 52.2 mmol) and isobutyl chloroformate (2.4 ml, 18.3 mmol) were added at ꢀ
158. After 15 min, a cold (08, bath temp.) soln. of the TFA salt in THF (27 ml) was added dropwise.
After 1 h at 08, the ice-bath was removed, and the soln. was allowed to warm to r.t. Stirring was continued
for 22 h. The mixture was then diluted with AcOEt and washed with 5% citric acid, sat. aq. NaHCO3, and
sat. aq. NaCl soln. All aq. layers were additionally extracted twice with AcOEt. The combined org. layers
were dried (MgSO4) and evaporated. Flash chromatography (FC; AcOEt/PE 1:1) yielded Boc-d-Val-d-
Ala-d-Leu-OBn (4.71 g, 52%). 1H-NMR Data in agreement with those in [20].
Boc-d-Leu-Aib-OBn. Boc-d-Leu-OH · H2O (4.92 g, 19.7 mmol) was dissolved in CH2Cl2 (36 ml) and
heated under reflux using an ꢂinverseꢃ water separator for 12 h and evaporated. To a soln. of the resulting
Boc-d-Leu-OH in THF (100 ml) at ꢀ 158, NMM (2.3 ml, 20.9 mmol), isobutyl chloroformate (2.75 ml,
21.0 mmol) were added, and, after stirring for 5 min, a cold soln. (08, ice bath) of Boc-Aib-OBn · TosOH
(7.42 g, 22.0 mmol) and NMM (2.42 ml, 22.0 mmol) in DMF (50 ml) was added. The mixture was allowed
to warm to r.t. within 2 h, and stirring was continued for 22 h. The soln. was evaporated under reduced
pressure, and the residue was dissolved in AcOEt, washed with 1n HCl (3ꢁ), sat. aq. K2CO3 (3ꢁ), H2O
(3ꢁ), and sat. aq. NaCl soln, and dried (MgSO4). Removal of the solvent under reduced pressure gave
Boc-d-Leu-Aib-OBn (7.23 g, 89%). For anal. purposes, a sample was recrystallized from CHCl3/hexane.
Colorless crystals. [a]D ¼ þ48.0 (c ¼ 1.0, CHCl3). IR (CHCl3): 3435m, 3007m, 2961m, 2871w, 1734s, 1681s,
1
1498s, 1455s, 1389m, 1368s, 1277m, 1159s, 1050m, 1023w, 871w, 656w, 630w, 619w. H-NMR (300 MHz,
CDCl3): 0.88 (d, J ¼ 6.2, Me); 0.89 (d, J ¼ 6.5, Me); 1.41 (s, t-Bu); 1.53 (s, Me); 1.54 (s, Me); 1.56 – 1.66
(m, 3 CH); 3.90 – 4.10 (m, CHN); 4.86 (d, J ¼ 8.1, NH); nA ¼ 5.11, nB ¼ 5.16 (AB, JAB ¼ 12.3, CH2O);
6.77 (br. s, NH); 7.32 – 7.37 (m, 5 arom. H). 13C-NMR (75 MHz, CDCl3): 22.0, 22.9, 24.7 (Me); 24.8 (CH);
28.3 (Me); 41.0 (CH2); 53.0 (CH); 56.4 (Me); 67.3 (CH2); 80.1 (C); 128.4, 128.5, 128.7 (CH); 136.0, 156.1,
167.8, 172.0, 174.4 (C). ESI-MS (pos. mode): 445 (25, [M þ K]þ), 429 (100, [M þ Na]þ), 407 (17, [M þ
H]þ). Anal. calc. for C22H34N2O5 (406.52): C 65.00, H 8.43, N 6.89; found: C 64.93, H 8.31, N 7.00.
Boc-d-Ala-d-Leu-Aib-OBn. Boc-d-Leu-Aib-OBn (5.48 g, 13.5 mmol) was Boc-deprotected in
CH2Cl2 (27 ml) according to GP 9 for 2 h. A soln. of the resulting TFA salt in CHCl3 (85 ml) was cooled
to 08 (ice-bath) and treated with Boc-d-Ala-OH (2.55 g, 13.5 mmol), NMM (4.16 ml, 37.8 mmol), HOBt
(2.45 g, 16.2 mmol), and EDC (2.58 g, 13.5 mmol). Stirring was continued for 2 h at 08 (ice bath) and at
r.t. for 22 h. The mixture was thoroughly washed with 1n HCl, aq. sat. NaHCO3, and NaCl soln. The org.
phase was dried (MgSO4) and then concentrated under reduced pressure to yield Boc-d-Ala-d-Leu-Aib-
OBn (5.6 g, 87%). For anal. purposes, a sample was recrystallized from CHCl3/hexane and further
purified by FC (MeOH/CH2Cl2 3 :97). White solid. M.p. 115 – 1188. Rf (MeOH/CH2Cl2 3 :97) 0.20.
[a]D ¼ þ62.6 (c ¼ 1.0, CHCl3). IR (CHCl3): 3678w, 3434m, 3008m, 2982m, 2872w, 1676s, 1603w, 1499s,
1455s, 1389m, 1368m, 1289m, 1159s, 1067w, 1027w, 852w, 658w, 628w. 1H-NMR (400 MHz, CDCl3): 0.87
(d, J ¼ 6.3, Me); 0.89 (d, J ¼ 6.5, Me); 1.31 (d, J ¼ 7.1, Me); 1.43 (s, t-Bu); 1.45 – 1.69 (m, 3 CH); 1.52 (s,
Me); 1.54 (s, Me); 4.07 – 4.11 (m, CHN); 4.35 – 4.44 (m, CHN); 5.04 (d, J ¼ 6.5, NH); 5.13 (s, CH2O);
6.56 (d, J ¼ 8.2, NH); 6.86 (s, NH); 7.29 – 7.37 (m, 5 arom. H). 13C-NMR (100 MHz, CDCl3): 17.9, 21.8,
22.9 (Me); 24.6 (CH); 24.7, 24.7, 28.2 (Me); 40.4 (CH2); 50.4, 51.6 (CH); 56.3 (C); 67.1 (CH2); 80.4 (C);
128.1, 128.2, 128.4 (CH); 135.7, 155.6, 170.9, 172.7, 174.0 (C). HR-MALDI-MS: 500.2729 (81, [M þ Na]þ,
C25H39N3NaOþ; calc. 500.2737), 400.2205 (100, [M ꢀ Boc þ Na]þ, C20H31N3NaOþ4 ; calc. 400.2212),
378.2392 (5, [M þ H ꢀ Boc]þ; C20H32N3O4þ ; calc. 378.2393).

Helvetica Chimica Acta – Vol. 92 (2009)
2711
Boc-d-Val-d-Ala-d-Leu-Aib-OBn. Boc-d-Ala-d-Leu-Aib-OBn (4.37 g, 9.15 mmol) was Boc-depro-
tected in CH2Cl2 (18.3 ml) according to GP 9 for 3 h. The resulting TFA salt was dissolved in CHCl3
(18.3 ml) and treated with Boc-d-Val-OH (1.99 g, 9.16 mmol, added as solid), NMM (2.82 ml,
25.6 mmol), HOBt (1.52 g, 10.0 mmol), and EDC (1.75 g, 9,13 mmol) according to GP 10a for 1 h at
08 (ice-bath). THF (3 ml) was added, and the mixture was allowed to warm to r.t. and then stirred for
2.5 d. The mixture was worked up as described in GP 10b. FC (AcOEt/PE 4 :1) yielded Boc-d-Val-d-Ala-
d-Leu-Aib-OBn (4.12 g, 78%). Colorless glass. Rf (AcOEt/PE 4 :1) 0.54. [a]D ¼ þ52.1 (c ¼ 1.1, CHCl3).
1
IR (CHCl3): 3430m, 3008m, 2963m, 1675s, 1498s, 1455m, 1389m, 1368m, 1278m, 1157s, 984w. H-NMR
(400 MHz, CDCl3): 0.85 (d, J ¼ 6.4, Me); 0.88 (d, J ¼ 6.5, Me); 0.92 (d, J ¼ 6.9, Me); 0.95 (d, J ¼ 6.9, Me);
1.35 (d, J ¼ 7.1, Me); 1.39 – 1.65 (m, 2 CH); 1.44 (s, t-Bu); 1.51 (s, Me); 1.53 (s, Me); 1.67 – 1.74 (m, CH);
2.09 – 2.15 (m, CH); 3.92 – 3.95 (m, CHN); 4.38 – 4.45 (m, 2 CHN); 5.10 – 5.16 (m, NH, CH2O); 6.82 (d,
J ¼ 6.5, NH); 6.88 (d, J ¼ 8.0, NH); 7.01 (br. s, NH); 7.28 – 7.36 (m, 5 arom. H). 13C-NMR (100 MHz,
CDCl3): 17.8, 18.2, 19.3, 21.7, 23.0, 24.7 (Me); 24.8 (CH); 24.9, 28.3 (Me); 30.6 (CH); 40.3 (CH2); 49.4,
51.7 (CH); 56.2 (C); 60.4 (CH); 66.9 (CH2); 80.6 (C); 128.1, 128.1, 128.5 (CH); 136.0, 156.3, 171.1, 171.8,
172.1, 174.1 (C). FAB-MS: 1154 (10), 1153 (13, [2M þ H]þ), 578 (29), 577 (100, [M þ H]þ), 469 (13), 384
(12), 328 (19), 307 (17), 215 (16), 194 (16), 115 (10). Anal. calc. for C30H48N4O7 (576.73): C 62.48, H 8.39,
N 9.71; found: C 62.35, H 8.24, N 9.56.
Boc-d-Val-d-Ala-d-Leu-Aib-OH. Boc-d-Val-d-Ala-d-Leu-Aib-OBn (2.01 g, 3.48 mmol) was trans-
formed in MeOH (83 ml) according to GP 8 for 23 h to yield Boc-d-Val-d-Ala-d-Leu-Aib-OH (1.65 g,
98%). Colorless glass. [a]D ¼ þ62.9 (c ¼ 1.1, CHCl3). IR (CHCl3): 3299m, 2966s, 1657s, 1509s, 1463m,
1
1456m, 1392m, 1368m, 1165s, 1092w, 1044w, 1017w, 869w. H-NMR (400 MHz, CDCl3): 0.87 – 0.95 (m,
4 Me); 1.32 (d, J ¼ 6.7, Me); 1.37 – 1.64 (m, 3 CH); 1.44 (s, t-Bu); 1.49 (s, Me); 1.53 (s, Me); 2.01 – 2.09 (m,
CH); 4.12 – 4.19 (m, CHN); 4.55 – 4.65 (m, CHN); 4.70 – 4.80 (m, CHN); 5.28 (d, J ¼ 8.6, NH); 7.35 (br. s,
NH); 7.76 (br. s, NH); 8.13 (d, J ¼ 6.8, NH); 13C-NMR (100 MHz, CDCl3): 17.8, 19.0, 19.2, 22.1, 22.8, 24.3
(Me); 24.6 (CH); 25.4, 28.2, 28.3 (Me); 31.7 (CH); 40.8 (CH2); 48.7, 51.6 (CH); 56.2 (C); 59.5 (CH); 80.2,
156.1, 171.6, 171.9, 172.9, 176.2 (C). FAB-MS: 995 (14, [2M þ Na]þ), 973 (29, [2M þ H]þ), 747 (13), 725
(23), 625 (16), 510 (15), 509 (53, [M þ Na]þ), 488 (30), 487 (100, [M þ H]þ), 469 (12), 431 (14), 387 (22),
384 (22), 328 (34), 217 (35), 215 (31), 171 (11), 155 (13), 154 (27), 138 (14), 137 (21), 136 (24), 116 (21),
111 (10), 106 (11), 103 (12).
Boc-d-Val-d-Ala-d-Leu-Aib-d-Val-d-Ala-d-Leu-OBn (1d). Boc-d-Val-d-Ala-d-Leu-OBn (1.57 g,
3.19 mmol) was Boc-deprotected in CHCl3 (6.4 ml) according to GP 9 for 2 h. A soln. of the resulting
TFA salt in CHCl3 (3.2 ml) at 08 (ice bath) was treated with NMM (0.98 ml, 8.89 mmol), a cold (08, ice-
bath) soln. of Boc-d-Val-d-Ala-d-Leu-Aib-OH (1.55 g, 3.19 mmol) in DMF (3.2 ml), HOBt (0.58 g,
3.83 mmol), and EDC (0.61 g, 3.18 mmol). The mixture was allowed to warm to r.t. After stirring for
27 h, the soln. was evaporated under reduced pressure, and the residue was dissolved in CHCl3 and
washed with 1n HCl, sat. aq. NaHCO3, and sat. aq. NaCl soln. All aq. layers were additionally extracted
with CHCl3. The combined org. layers were dried (MgSO4) and concentrated. FC (MeOH/CH2Cl2 1:9)
yielded 1d (2.18 g, 79%). Yellowish crystalline solid. M.p. 179 – 1818. Rf (MeOH/CH2Cl2 1:9) 0.59.
[a]D ¼ þ17.0 (c ¼ 1.1, CHCl3). CD (0.2 mm in MeOH): þ 7.8 ꢁ 104 (200 nm). IR (CHCl3): 3326m, 2963m,
2873w, 1665s, 1526s, 1369w, 1156m, 984w. 1H-NMR (400 MHz, CDCl3): 0.87 – 1.04 (m, 8 Me); 1.43 – 1.52
(m, 4 Me); 1.48 (s, t-Bu); 1.53 – 1.81 (m, 5 CH); 2.09 – 2.16 (m, 2 CH); 2.42 – 2.50 (m, CH); 3.70 – 3.73 (m,
CHN); 4.08 – 4.19 (m, 3 CHN); 4.48 – 4.63 (m, 2 CHN); nA ¼ 5.14, nB ¼ 5.16 (AB, JAB 12.5, CH2O); 5.66
(d, J ¼ 2.7, NH); 6.93 (d, J ¼ 6.7, NH); 7.03 (d, J ¼ 3.8, NH); 7.26 – 7.39 (m, 2 NH, 5 arom. H); 7.54 (s, NH);
7.69 (d, J ¼ 7.9, NH). 13C-NMR (100 MHz, CDCl3): 17.1, 17.3, 17.3, 18.8, 19.3, 19.4, 21.6, 21.7, 22.6, 22.8,
23.2 (Me); 24.5, 24.8 (CH); 27.1, 28.2 (Me); 28.8, 29.6 (CH); 40.1, 40.6 (CH2); 49.4, 50.9, 51.5, 54.0 (CH);
56.9 (C); 60.1, 62.2 (CH); 66.5 (CH2); 81.3 (C); 127.8, 127.9, 128.1, 128.4 (CH); 136.0, 157.4, 171.5, 172.5,
173.1, 173.3, 173.4, 173.8, 176.1 (C). FAB-MS: 1058 (121), 883 (20, [M þ Na]þ), 882 (40), 862 (12), 861
(48, [M þ H]þ), 861 (100, Mþ), 766 (10), 640 (19), 639 (57), 569 (22), 568 (66), 512 (14), 470 (14), 469
(48), 414 (14), 413 (59), 384 (10), 369 (10), 328 (37), 293 (10), 215 (14).
TFA · H-d-Val-d-Ala-d-Leu-Aib-d-Val-d-Ala-d-Leu-OBn (1c). Compound 1d (0.30 g, 0.35 mmol)
was Boc-deprotected in CH2Cl2 (0.7 ml) according to GP 9 for 2 h. The residue was co-evaporated with
toluene and CHCl3, and dried under h.v. to yield quantitatively 1c (0.35 g). Colorless glass. [a]D ¼ þ27.6
(c ¼ 1.1, CHCl3). IR (CHCl3): 3315m, 2963m, 1737m, 1664s, 1535m, 1469w, 1386w, 1172s, 1038w. 1H-NMR

2712
Helvetica Chimica Acta – Vol. 92 (2009)

Helvetica Chimica Acta – Vol. 92 (2009)
2713
Table 2. NOEs for Chimeric b/a-Tridecapeptide 4
Residue
H-Atom
Residue
H-Atom
dNOE [ꢄ]
1
2
2
2
3
3
3
3
3
3
4
4
4
4
4
5
5
5
6
6
6
6
6
7
7
7
8
8
8
9
9
11
11
11
12
12
13
13
13
2
3
3
5
4
g
1
2
2
2
3
3
3
3
3
3
4
4
4
4
4
5
5
5
6
6
6
6
6
7
7
7
8
8
8
9
9
11
11
11
12
12
13
13
13
3
4
4
4
3
b
2.5
3.5
3.2
2.9
2.9
2.7
2.9
2.9
3.1
2.9
2.6
2.8
2.9
3.3
2.8
2.6
3.0
3.6
2.5
2.7
2.2
2.9
2.9
2.9
2.7
1.8
2.8
2.0
3.0
2.7
2.7
3.1
2.9
2.6
3.0
3.6
2.8
2.9
3.3
2.4
2.5
3.6
3.0
2.7
3.8
3.8
4.4
4.3
2.7
b
g1
g2
b
HN
HN
aRe
aSi
g*a)
HN
HN
aSi
b
HN
b
b
b
d
HN
aSi
b
g*
b
HN
HN
aRe
b
g*
HN
aSi
HN
b
b
e*
b
aSi
aRe
b
HN
g
b
HN
HN
b*
HN
bRe
a
g
a
a
bSi
HN
bRe
bSi
HN
HN
a
bSi
HN
a
g
H2
b*
a
HN
HN
HN
a
a
a
b
b*
a
HN
HN
HN
HN
HN
aSi
aSi
HN
b
b*
aRe
aRe
b
HN
HN
HN
HN
HN
HN
HN
4
5
5
5
3
4
4
6
HN
HN
aSi
7
6

2714
Helvetica Chimica Acta – Vol. 92 (2009)
Table 2 (cont.)
Residue
H-Atom
Residue
H-Atom
dNOE [ꢄ]
6
7
6
7
8
7
9
8
10
9
aRe
HN
HN
a
7
6
5
8
7
8
8
9
9
10
10
11
11
12
12
4
5
6
7
7
HN
b
2.6
3.3
2.3
2.4
3.4
3.0
3.5
3.7
2.5
4.6
3.0
2.9
3.6
3.3
2.8
4.1
2.6
3.2
2.9
3.7
3.4
3.3
3.2
4.2
4.2
4.0
3.7
4.5
3.8
3.5
4.1
3.0
3.9
3.8
2.8
3.9
4.7
4.8
3.5
aRe
HN
b*
HN
b*
HN
a
HN
HN
HN
HN
HN
g
HN
HN
HN
a
9
HN
HN
HN
HN
HN
g
10
12
13
13
1
2
3
4
4
4
4
6
9
8
10
8
8
9
b
HN
b
aRe
HN
aRe
g*
HN
HN
b
b
b
a
a
6
7
3
7
b
a
aSi
a
HN
bRe
10
8
11
4
11
11
8
13
13
13
13
9
HN
bSi
H2
g*
H2
HN
a
HN
bSi
HN
a
9
a
10
11
11
11
11
11
11
13
13
HN
a
HN
HN
HN
HN
a
bRe
bSi
HN
HN(1)
HN(1)
b*
13
9
9
b*
HN
a
HN
a) *: Pseudoatom used for calculations.
(400 MHz, CD3OD): 0.87 (d, J ¼ 6.2, Me); 0.90 – 0.97 (m, 5 Me); 1.03 (d, J ¼ 6.9, Me); 1.05 (d, J ¼ 7.0,
Me); 1.35 (d, J ¼ 7.2, Me); 1.38 (d, J ¼ 7.1, Me); 1.44 (s, Me); 1.46 (s, Me); 1.51 – 1.78 (m, 6 CH); 2.14 – 2.34
(m, 2 CH); 3.65 – 3.70 (m, CHN); 4.10 – 4.13 (m, CHN); 4.16 – 4.23 (m, CHN); 4.33 – 4.38 (m, CHN);
4.41 – 4.49 (m, 2 CHN); nA ¼ 5.13, nB ¼ 5.15 (AB, JAB 12.3, CH2O); 7.28 – 7.38 (m, 5 arom. H). 13C-NMR
(100 MHz, CD3OD): 17.9, 18.0, 18.1, 18.9, 19.8, 21.9, 22.2, 23.3, 23.3, 25.4 (Me); 25.8, 31.3, 31.6 (CH); 41.3,
41.5 (CH2); 50.3, 50.4, 52.4, 54.3 (CH); 58.0 (C); 59.5, 60.7 (CH); 67.9 (CH2); 129.3, 129.3, 129.6 (CH);

Helvetica Chimica Acta – Vol. 92 (2009)
2715
Table 3. 1H- and 13C-NMR Data and Assignments for Acetyl-b-heptapeptide 5b. d in ppm, J in Hz.
Residue NH CH2(a) HꢀC(b)
HꢀC(g), Me(d), CH2(e/z), C(a) C(b) C(g) C(d)
C(e/z)
(3J(HN,Hb)) Me(g), CH2(d) Me(e)
CH2(g)
b3hVal1 8.04 2.52
b3hAla2 8.06 2.61
4.22 (9.60) 1.79
0.94
1.39
39.18 53.40 34.06 19.41
43.63 44.14 21.30
1.65/2.92 41.76 47.23 35.95 23.78
4.38 (NA)
b3hLys3 8.07 2.35/2.56 4.33 (NA)
1.14
1.52
28.40/41.0
b3hIle4 7.79 2.37
4.28 (8.95) 1.53
0.91/1.11 0.926
38.80 52.27 40.93 15.7/27.17 19.64
b3hAla5 7.46 2.30/2.40 4.47 (8.52) 1.15
43.43 43.93 21.32
38.41 49.62 64.80
NA 49.03 41.17
b3hSer6 7.63 2.32/2.50 4.36 (8.58) 3.43/3.47
b3hTyr7 7.97 2.47/2.58 4.49 (8.51)
2.64/2.72
Table 4. NOEs for N-Acetyl-b-heptapeptide 5b
Residue
H-Atom
Residue
H-Atom
dNOE [ꢄ]
2
2
3
3
4
4
4
5
5
6
6
2
5
5
6
7
1
2
3
5
5
5
5
b
2
2
3
3
4
4
4
5
g
3.1
2.9
2.9
2.9
2.7
2.9
2.9
2.8
2.5
2.7
2.9
3.5
3.9
3.9
4.0
3.1
3.4
3.0
3.4
3.1
2.9
3.6
3.7
b
HN
g*
HN
e1
b
b
b
b
e2
b
HN
HN
HN
g1
HN
a*
HN
HN
HN
aSi
b
b
g
5
6
6
b
b
HN
b
ACE1
6
6
7
6
4
5
6
7
8
8
8
HN
b
HN
a*a)
g
b
HN
HN
g
b
b
b
g
g1
g2
g
a) * ¼ Pseudoatom used for calculations.
137.3, 169.3, 173.3, 173.6, 174.7, 174.8, 175.1, 177.2, 177.2 (C). FAB-MS: 1521 (10), 98 1 (34), 959 (16), 784
(29), 783 (72), 782 (86, [M þ Na]þ), 763 (10), 762 (36), 761 (82), 760 (100, [M þ H]þ), 369 (23), 284 (19),
171 (17).
Boc-d-Val-d-Ala-d-Leu-Aib-d-Val-d-Ala-d-Leu-OH (1b). Compound 1c (0.20 g, 0.23 mmol) was
transformed in MeOH (5.6 ml) according to GP 8 for 18 h to yield 1b (0.15 g, 85%). Colorless crystalline
solid. M.p. 142 – 1448. [a]D ¼ þ41.9 (c ¼ 0.9, MeOH). CD (0.2 mm in MeOH): þ 5.9 ꢁ 104 (201 nm). IR
(KBr): 3320m, 2962m, 2873w, 1654s, 1528s, 1469m, 1388m, 1367m, 1247m, 1168m, 1044w, 668w. 1H-NMR
(400 MHz, CD3OD): 0.89 – 4.98 (m, 8 Me); 1.37 (d, J ¼ 7.2, Me); 1.42 (d, J ¼ 7.2, Me); 1.46 (s, Me); 1.48 (s,

2716
Helvetica Chimica Acta – Vol. 92 (2009)
Table 5. 1H- and 13C-NMR Data and Assignments for PEG-b-heptapeptide 5c. d in ppm, J in Hz.
Residue NH CH2(a) HꢀC(b) HꢀC(g), Me(d), CH2(e/z), C(a) C(b) C(g) C(d)
(3J(HN,Hb)) Me(g), CH2(d) Me(e)
CH2(g)
C(e/z)
b3hVal1 8.14 2.40/2.55 4.25 (9.20) 1.80
b3hAla2 8.12 2.36/2.66 4.39 (9.24) 1.14
b3hLys3 8.09 2.36/2.57 4.34 (8.89) 1.52
0.95
38.80 53.24 34.25 19.54
43.55 43.92 21.02
1.64/1.75 41.66 46.91 35.90 23.58
1.40
0.93/1.11 0.928
28.31/41.04
b3hIle4 7.79 2.37
4.29 (8.95) 1.53
38.75 52.32 41.07 15.72/27.24 19.70
b3hAla5 7.42 2.29/2.41 4.49 (8.88) 1.15
b3hSer6 7.62 2.32/2.52 4.38 (8.66) 3.42/3.48
b3hTyr7 7.80 2.44/2.57 4.50 (9.02) 2.64/2.72
43.32 43.80 21.17
38.37 49.55 64.90
39.12 48.80 41.14
t-Bu); 1.49 (s, Me); 1.56 – 1.77 (m, 6 CH); 1.99 – 2.08 (m, CH); 2.21 – 2.29 (m, CH); 3.78 – 3.82 (m, CHN);
4.12 – 4.16 (m, CHN); 4.19 – 4.25 (m, 2 CHN); 4.35 – 4.46 (m, 2 CHN); 6.81 (d, J ¼ 5.7, NH); 7.34 (d, J ¼
7.4, NH); 7.79 (d, J ¼ 6.7, NH); 7.87 – 7.96 (m, 3 NH); 8.31 (d, J ¼ 5.2, NH). 13C-NMR (100 MHz,
CD3OD): 17.5, 18.0, 18.6, 19.0, 19.6, 19.8, 21.8, 22.2, 23.2, 23.5, 25.0 (Me); 25.8, 25.9 (CH); 26.3, 28.8
(Me); 31.1, 31.6 (CH); 41.4, 41.7 (CH2); 50.6, 51.6, 52.2, 54.6 (CH); 58.1 (C); 60.9, 61.0, 62.7 (CH); 81.1,
158.8, 173.4, 174.9, 175.0, 175.7, 175.8, 177.6 (C). FAB-MS: 1562 (5, [2M þ Na]þ), 990 (16), 793 (41), 792
(100, [M þ Na]þ), 639 (11), 568 (13), 469 (11), 413 (17), 328 (12).
TFA · H-d-Val-d-Ala-d-Leu-Aib-d-Val-d-Ala-d-Leu-OH (1a). Compound 1b (95 mg, 0.12 mmol)
was Boc-deprotected in CHCl3 (0.25 ml) according to GP 9 for 2 h. The residue was co-evaporated with
toluene, dissolved in TFE, filtered, and concentrated under reduced pressure to yield 1a (73 mg, 88%).
Colorless glass. [a]D ¼ þ40.8 (c ¼ 0.5, MeOH). CD (0.2 mm in MeOH): þ 4.3 ꢁ 104 (198 nm). IR (KBr):
3315m, 2964m, 1659s, 1540s, 1469m, 1388m, 1203s, 800w, 722w. 1H-NMR (400 MHz, CD3OD): 0.87 – 0.99
(m, 6 Me); 1.04 (d, J ¼ 6.9, Me); 1.06 (d, J ¼ 6.9, Me); 1.38 (d, J ¼ 7.1, Me); 1.39 (d, J ¼ 7.2, Me); 1.44 (s,
Me); 1.46 (s, Me); 1.51 – 1.79 (m, 6 CH); 2.12 – 2.28 (m, 2 CH); 3.65 – 3.69 (m, CHN); 4.13 – 4.18 (m,
CHN); 4.20 – 4.25 (m, CHN); 4.35 – 4.47 (m, 3 CHN); 7.33 (2d, J ¼ 7.9, 2 NH); 7.98 (d, J ¼ 8.1, NH); 8.07
(d, J ¼ 7.3, NH); 8.22 – 8.23 (m, NH). 13C-NMR (100 MHz, CD3OD): 17.9, 18.0, 18.1, 18.9, 19.9, 21.9, 22.2,
23.3, 23.5, 25.6 (Me); 25.8, 25.9, 31.5, 31.6 (CH); 41.5, 41.6 (CH2); 50.3, 50.4, 52.1, 54.2 (CH); 58.1 (C);
59.6, 60.7 (CH); 169.3, 173.3, 174.6, 174.7, 174.9, 175.7, 177.1, 177.2 (C). FAB-MS: 715 (12), 714 (13), 694
(28), 693 (69), 692 (100, [M þ Na]þ), 671 (11), 670 (13, [M þ H]þ).
Preparation of 3. Boc-(R)-b3-hVal-(S)-b3-hAla-(S)-b3-hLeu-(R)-b3-hVal-(S)-b3-Ala-(S)-b3-Leu-d-
Val-d-Ala-d-Leu-Aib-d-Val-d-Ala-d-Leu-OBn. A soln. of 1c (0.13 g, 0.15 mmol) in CHCl3/1,2-dime-
thoxyethane (DME) 1:1 (1.2 ml) at 08 (ice-bath) was subsequently treated with Et3N (83 ml, 0.60 mmol),
BocTFA · H-(R)-b3-hVal-(S)-b3-hAla-(S)-b3-hLeu-(R)-b3-hVal-(S)-b3-Ala-(S)-b3-Leu-OH [1][21]
(0.12 g, 0.15 mmol), HOBt (28 mg, 0.19 mmol), and EDC (37 mg, 0.19 mmol). The mixture was allowed
to warm to r.t. After 14 h, CHCl3 (1 ml) was added to the mixture, and stirring was continued for another
10 h. The mixture was then evaporated under reduced pressure and dried under h.v. The residue was
dissolved in MeOH under reflux, filtered, and cooled to 48 (cooling room). After stirring for 2 h at 48
(cooling room), the precipitate was filtered and dried under h.v. to yield Boc-(R)-b3-hVal-(S)-b3-hAla-
(S)-b3-hLeu-(R)-b3-hVal-(S)-b3-Ala-(S)-b3-Leu-d-Val-d-Ala-d-Leu-Aib-d-Val-d-Ala-d-Leu-OBn
(96 mg, 43%). White amorphous solid. M.p. 2498 (dec.). [a]D ¼ þ26.9 (c ¼ 0.6, 2,2,2-trifluoroethanol
(TFE)). CD (0.2 mm in MeOH): ꢀ 0.99 ꢁ 104 (218 nm), 0 (212 nm), þ 8.5 ꢁ 104 (199 nm). IR (KBr):
3304s, 3067w, 2960m, 2872w, 1652s, 1538s, 1457m, 1368m, 1249m, 1172m, 698w. 1H-NMR (CD3OD/CDCl3
1:3): 0.88 – 0.93 (m, 12 Me); 0.95 – 1.03 (m, 4 Me); 1.17 (d, J ¼ 6.7, 2 Me); 1.28 – 1.37 (m, 2 CH, 2 Me);
1.40 – 1.51 (m, 6 CH); 1.44 (s, t-Bu); 1.47 (s, Me); 1.51 (s, Me); 1.53 – 1.83 (m, 8 CH); 2.12 – 2.55 (m,
6 CH2); 3.72 – 3.80 (m, CHN); 4.01 (d, J ¼ 6.5, CHN); 4.07 – 4.12 (m, 2 CHN); 4.14 – 4.30 (m, 6 CHN);
4.38 (q, J ¼ 7.3, CHN); 4.49 – 4.52 (m, CHN); 5.16 (s, CH2O); 6.17 (d, J ¼ 10.1, NH); 6.95 (d, J ¼ 7.6, NH);
7.28 – 7.38 (m, 5 arom. H); 7.71 (d, J ¼ 2.0, NH); 8.08 (d, J ¼ 7.84, NH). 13C-NMR (CD3OD/CDCl3 1:3):
17.1, 17.8, 18.3, 18.4, 19.1, 19.6, 19.6, 19.7, 20.1, 20.5, 21.8, 22.1, 22.1, 22.3, 23.0, 23.2, 23.5, 23.7, 23.7, 24.3

Helvetica Chimica Acta – Vol. 92 (2009)
Table 6. NOEs for PEG-b-Heptapeptide 5c
2717
Residue
H-Atom
Residue
H-Atom
dNOE [ꢄ]
2
2
2
2
3
3
4
4
5
5
6
6
7
7
8
8
8
2
2
2
5
5
7
7
g
2
2
2
2
3
3
4
4
5
5
6
6
7
7
8
8
aRe
aSi
3.2
3.5
2.9
3.2
3.0
3.0
3.2
2.9
3.0
3.0
2.8
2.9
2.8
2.9
2.9
2.7
2.9
3.0
2.9
4.0
4.2
3.7
3.1
4.2
4.4
2.3
3.1
3.2
5.0
4.9
3.3
4.3
4.4
4.9
3.4
3.2
3.4
3.0
g
b
HN
HN
HN
g*a)
e1
HN
HN
HN
g*
HN
g1
HN
HN
aRe
g1
g
b
b
b
b
b
g
b
b
b
b
b
HN
HN
HN
HN
HN
b
8
PEG1
PEG1
PEG1
6
6
6
6
7
a1
a2
b*
b
HN
HN
HN
HN
HN
a1
a2
b
HN
A1
b
6
8
HN
aRe
b
7
4
4
PEG1
PEG1
b
3
3
2
4
4
4
5
6
7
8
PEG1
PEG1
5
PEG1
PEG1
7
2
3
5
5
HX19
HX20
b
b
g
e1
e2
g*
b
a1
a2
g1
aRe
HN
HN
g
b
b
b
a) * ¼ Pseudoatom used for calculations HX19, HX120 protons next to terminal NH2 in PEG.
(Me); 25.4, 25.7, 25.8 (CH); 26.7, 27.0, 28.8 (Me); 30.1 (CH); 30.4 (CH2); 30.8, 32.7, 32.8, 33.4 (CH); 40.0,
40.1, 40.7, 41.0 (CH2); 43.0, 43.3, 43.8, 44.2 (CH2); 44.2 (CH); 44.3 (CH2); 46.2, 46.5, 50.4, 51.7, 52.0, 52.9,
54.6, 54.8 (CH); 57.6 (C); 61.0, 61.6 (CH); 67.5 (CH2); 79.8, 128.3, 128.9, 128.9, 129.2, 135.7 (CH); 136.9,
157.6, 172.1, 172.2, 172.3, 172.4, 172.7, 173.1, 173.2, 173.8, 174.3, 174.7, 174.8, 175.7, 177.4 (C). FAB-MS:
1534 (24), 1533 (100, [M þ Na]þ), 1412 (12), 1411 (48).
TFA · H-(R)-b3-hVal-(S)-b3-hAla-(S)-b3-hLeu-(R)-b3-hVal-(S)-b3-Ala-(S)-b3-Leu-d-Val-d-Ala-d-
Leu-Aib-d-Val-d-Ala-d-Leu-OH (3). Boc-(R)-b3-hVal-(S)-b3-hAla-(S)-b3-hLeu-(R)-b3-hVal-(S)-b3-
Ala-(S)-b3-Leu-d-Val-d-Ala-d-Leu-Aib-d-Val-d-Ala-d-Leu-OBn (61 mg, 40 mmol) was transformed in

2718
Helvetica Chimica Acta – Vol. 92 (2009)
Table 7. 1H- and 13C-NMR Data and Assignments for DPA b-Peptide 6. d in ppm, J in Hz.
Residue NH CH2(a) HꢀC(b)
HꢀC(g), HꢀC(d), CH2(e/z), C(a) C(b) C(g) C(d) CH2(e/z)
(3J(HN,Hb)) Me(g),
Me(d),
CH2(d)
Me(e)
CH2(g)
b3hVal1 9.04 2.63/2.78 4.48
1.96
1.10
1.23/1.36 1.51
1.60
1.80/1.88 2.87
3.56
1.16
1.54
NA
1.78
1.01
39.89 54.31 34.28 19.74
43.52 44.12 21.72
42.60 52.65 46.55 26.05 23.42
37.71 52.21 40.45 27.20 19.59
NA 47.33 32.61 41.92
38.32 49.98 65.28
b3hAla2 8.25 2.22
b3hLeu3 8.06 2.45
4.31 (8.44)
4.27 (8.95)
0.83
b3hIle4 8.14 2.40/2.66 4.40 (9.10)
1.12/1.50 0.902
b3hGlu5 8.16 2.46
4.51 (NA)
b3hSer6 8.28 2.492.78 4.51
b3hAla7 8.42 2.41/2.61 4.60 (8.80)
b3hLys8 7.45 2.30/2.51 4.47 (9.10)
b3hAsp9 7.62 2.49/2.61 4.88 (9.02)
b3hVal10 7.86 2.50/2.64 4.26 9.12
43.35 43.72 21.49
1.46
0.93
1.65/2.96 41.92 47.73 36.45 24.65 28.92/41.13
40.17 44.57
41.84 46.08 33.86 1.35
TFE (1 ml) according to GP 8 for 18 h. The residue was Boc-deprotected with TFA (2 ml) according to
GP 9 for 2 h. The crude product was co-evaporated with toluene, dissolved in TFE, filtered, and
evaporated under reduced pressure to yield 3 (62 mg, quant.). Colorless glass. [a]D ¼þ 21.5 (c ¼ 1.1,
TFE). CD (0.2 mm in MeOH): þ 7.9 ꢁ 104 (204 nm). IR (KBr): 3304m, 2963m, 1652s, 1540s, 1469m,
1387m, 1202s, 1173s, 800w , 706w. 1H-NMR (400 MHz, CD3OD): 0.89 – 0.98 (m, 14 Me); 1.06 (d, J ¼ 6.8,
Me); 1.07 (d, J ¼ 6.9, Me); 1.14 (d, J ¼ 6.7, Me); 1.18 (d, J ¼ 6.7, Me); 1.22 – 1.49 (m, 4 CH); 1.37 (d, J ¼
7.2, Me); 1.41 (d, J ¼ 7.2, Me); 1.45 (s, Me); 1.48 (s, Me); 1.55 – 1.79 (m, 9 CH); 1.98 – 2.06 (m, CH);
2.08 – 2.18 (m, CH); 2.20 – 2.27 (m, CH); 2.34 – 2.62 (m, 6 CH2); 3.43 – 3.48 (m, CHN); 4.12 – 4.22 (m,
4 CHN); 4.27 – 4.46 (m, 7 CHN); 7.35 (d, J ¼ 7.6, NH); 7.74 (d, J ¼ 9.5, NH); 7.86 – 7.90 (m, NH); 7.99 –
8.09 (m, 3 NH); 8.19 (d, J ¼ 6.0, NH). 13C-NMR (100 MHz, CD3OD): 17.8, 18.1, 18.2, 18.8, 18.9, 19.0,
19.7, 19.9, 20.7, 20.9, 21.9, 22.3, 22.6, 22.7, 23.3, 23.5, 23.6, 23.8, 25.2 (Me); 25.8, 25.9, 26.0, 31.2, 31.6, 32.0,
33.6 (CH); 35.9, 39.4, 41.3, 41.6, 42.4, 42.8, 43.3 (CH2); 43.9, 44.0 (CH); 45.1, 45.7 (CH2); 46.0, 46.3, 50.6,
50.9, 52.1, 53.0, 54.6, 56.2 (CH); 58.1 (C); 60.7, 60.9 (CH); 171.6, 172.0, 172.2, 172.6, 172.8, 173.4, 173.6,
173.8, 174.8, 175.0, 175.5, 175.6, 177.4 (C). FAB-MS: 1366 (17), 1365 (17), 1360 (16, [M þ K]þ), 1358
(32), 1348 (11), 1345 (16), 1344 (38, [M þ Na]þ), 1343 (94), 1342 (57), 1329 (11), 1327 (15), 1324 (18),
1323 (14), 1321 (100, Mþ), 1320 (43), 750 (19), 439 (11), 411 (16), 397(14).
H-(R)-b3hVal-(S)-b3hGlu-(S)-b3hLeu-(S)-b3hAla-(S)-b3hLys-(R)-b3hIle-(R)-Phe-(R)-Ser-(R)-
Leu-Aib-(R)-Trp-(R)-Thr-(R)-Ala-NH2 (4). Rink-Amide AM resin (100 mg, 0.071 mmol, 0.71 mmol/g)
was loaded with Fmoc-d-Ala-OH according to GP 2. SPPS was continued according to GP 5, until all
remaining residues were incorporated. For couplings of Fmoc-Aib-OH and Fmoc-(S)-b3hLys(Boc)-OH,
the coupling time was prolonged to 2 h. After final Fmoc deprotection (GP 4), the resin was dried under
h.v. for 2 h, and the peptide was cleaved from the resin according to GP 6 to give 129 mg of crude peptide.
A portion (44 mg) was purified by prep. RP-HPLC (34 – 95% A in 50 min: 5 min 34% A; 45 min. 40% A;
50 min. 95% A; 17 ml/min; tR 18.82 min) according to GP 7 to give 4 (16.9 mg) as the TFA salt. White
solid. Anal. RP-HPLC (5 – 99% A in 60 min: 45 min 50% A; 60 min 99% A): tR 37.45 min, purity > 98%.
1H- and 13C-NMR spectra: see Tables 1 and 2. MALDI-HRMS: 1545.9452 ([M þ H]þ, C77H124N16Oþ17
;
calc. 1545.9403).
H-(R)-b3hVal-(S)-b3hAla-(S)-b3hLys-(S)-b3hIle-(R)-b3hSer-(S)-b3hTyr-OH (5a). Wang resin
(300 mg, 1.1 mmol/g, 0.33 mmol) was esterified with Fmoc-b3-hTyr(tBu)-OH (625 mg, 1.32 mmol)
according to GP 1. Loading was determined to be 83% corresponding to 270 mmol of resin-bound Fmoc-
b3hTyr. After capping (GP 3) and Fmoc-deprotection (GP 4), the peptide – resin was coupled with
Fmoc-b3hSer(tBu)-OH (326 mg, 823 mmol), Fmoc-b3hAla-OH (268 mg, 823 mmol), Fmoc-b3hIle-OH
(302 mg, 823 mmol), Fmoc-b3hLys(Boc)-OH (397 mg, 823 mmol), Fmoc-b3hAla-OH (268 mg,
823 mmol), and Fmoc-b3hVal-OH (291 mg, 823 mmol) according to GP 5. After final Fmoc deprotection
(GP 4), the resin was washed with CH2Cl2 and dried under h.v. for 4 h. A portion of the resin (200 mg)

Helvetica Chimica Acta – Vol. 92 (2009)
Table 8. NOEs for DPA b-Peptide 6
2719
Residue
H-Atom
Residue
H-Atom
dNOE [ꢄ]
1
1
1
2
2
3
3
3
3
4
4
5
5
6
6
7
7
8
8
10
10
10
1
3
5
6
7
1
1
1
2
3
3
3
4
5
5
5
7
7
8
8
b
1
1
1
2
2
3
3
3
3
4
4
5
5
6
6
7
7
8
8
10
10
10
2
4
6
7
8
4
3
4
4
6
6
6
7
g
2.5
2.9
3.0
2.9
2.9
2.8
2.7
3.1
2.8
3.0
3.4
2.8
2.9
2.8
2.9
2.7
2.9
3.0
4.1
2.3
2.9
3.0
5.2
4.8
2.7
4.0
4.4
3.2
3.9
3.9
3.6
5.1
3.7
3.1
3.2
3.1
5.1
3.1
3.1
3.0
3.6
3.7
b
HN
HN
g*a)
HN
g1
g2
HN
d
g
b
b
b
b
b
b
b
HN
HN
g1
HN
g*
HN
g*
HN
HN
HN
g
g
b
b
b
b
b
b
b
d*
b
b
HN
g
g
HN
d
HN
HN
HN
b
d*
g*
HN
g
HN
b
HN
HN
HN
d
b
b
b
g*
b
g1
HN
g
b
b
b
2
3
8
4
10
10
10
HN
HN
b
d*
d*
b
HN
b
HN
HN
HN
b
b
a) * ¼ Pseudoatom used for calculations.
was then placed in a SPS reactor, and the peptide was cleaved from the resin according to GP 6 to give
94 mg of crude peptide. A portion of the crude peptide (50 mg) was purified by prep. RP-HPLC (20% A
in 5 min, 35 – 95% A in 30 min, C18) according to GP 7 to give 5a (16.2 mg) as a TFA salt. Colorless
solid. Anal. HPLC (5% A in 5 min, 5 – 95% A in 30 min, tR 26.36). Purity > 95%. MALDI-MS: 887 (10,

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Helvetica Chimica Acta – Vol. 92 (2009)
[M þ K]þ), 871 (14, [M þ Na]þ), 851 (12), 850 (48), 849 (100, [M þ H]þ). HR-MS: 849.5459
(C42H73N8NaOþ10 ; calc. 849.5450).
Ac-(R)-b3hVal-(S)-b3hAla-(S)-b3hLys-(S)-b3hIle-(R)-b3hSer-(S)-b3hTyr-OH (5b). A third portion
of peptide – resin was placed in a SPS reactor and acetylated according to GP 3. The peptide – resin was
then dried under h.v. for 2 h, and the peptide was cleaved from the resin according to GP 6a to give 37 mg
of crude peptide. Purification by prep. RP-HPLC (20% A in 5 min, 20 – 80% A in 30 min, C18) according
to GP 7 gave 5b (12.1 mg) as a TFA salt. Colorless solid. Anal. RP-HPLC (5% A in 5 min, 5 – 95% A in
30 min, tR 19.33). Purity > 95%. 1H- and 13C-NMR spectra: see Tables 3 and 4. MALDI-MS: 930.5 (12),
929.5 (29, [M þ K]þ), 915.5 (16), 914.5 (51), 913.5 (100, [M þ Na]þ), 892.6 (24), 891.6 (45, [M þ H]þ).
HR-MS: 913.5373 (C44H74N8NaOþ11 ; calc. 913.5375).
Amino-PEG6-(R)-b3hVal-(S)-b3hAla-(S)-b3hLys-(S)-b3hIle-(R)-b3hSer-(S)-b3hTyr-OH (5c). A
second portion of the peptide – resin (100 mg) was swollen in a SPS reactor (DMF, 4 ml, 30 min) and
then washed with DMF (5 ml, 3 ꢁ 1 min). The resin was filtered, and a soln. of Fmoc-amido dPEG6 acid
(41 mg, 70 mmol), HATU (26 mg, 68 mmol), and DIPEA (24 ml, 140 mmol) in DMF (3 ml) was added,
and the soln. was mixed by N2 bubbling for 3 h. The resin was filtered and washed with DMF (5 ml, 6 ꢁ
1 min). After final Fmoc deprotection (GP 4), the resin was dried under h.v. for 2 h, and the peptide was
cleaved from the resin according to GP 6 to give 53 mg of crude peptide. Purification by prep. RP-HPLC
(20% A in 5 min, 20 – 80% A in 30 min, C18) according to GP 7 gave 5c (11.1 mg) as a TFA salt. Colorless
1
solid. Anal. HPLC (5% A in 5 min, 5 – 95% A in 30 min, tR 18.98). Purity > 95%. H- and 13C-NMR
spectra: see Tables 5 and 6. MALDI-MS: 1223 (26, [M þ K]þ), 1208 (48), 1207 (72, [M þ Na]þ), 1187
(66), 1186 (67), 1185 (100, [M þ H]þ). HR-MS: 1184.7394 (C57H102N9O1þ7 ; calc. 1184.7402).
DPA-(R)-b3hVal-(S)-b3hAla-(S)-b3hLeu-(S)-b3hIle-(R)-b3hSer-(S)-b3hAla-(S)-b3hLys-(S)-
b3hAsp-(R)-b3hVal-OH (6). Wang resin (100 mg, 1.1 mmol/g, 0.11 mmol) was esterified with Fmoc-b3-
hVal-OH (194 mg, 550 mmol) according to GP 1. Loading was determined to be 86% corresponding to
95 mmol of resin-bound Fmoc-b3hVal. After capping (GP 3) and Fmoc-deprotection (GP 4), the
peptide – resin was coupled with Fmoc-b3hAsp(tBu)-OH (121 mg, 284 mmol), Fmoc-b3hLys(Boc)-OH
(137 mg, 284 mmol), Fmoc-b3hAla-OH (93 mg, 284 mmol), Fmoc-b3hSer(tBu)-OH (113 mg, 284 mmol),
Fmoc-b3hGlu(tBu)-OH (125 mg, 284 mmol), Fmoc-b3hIle-OH (104 mg, 284 mmol), Fmoc-b3hLeu-OH
(104 mg, 284 mmol), Fmoc-b3hAla-OH (93 mg, 284 mmol), Fmoc-b3hVal-OH (100 mg, 284 mmol), and
pyridine-2,6-dicarboxylic acid monobenzyl ester [17] (73 mg, 284 mmol) according to GP 5. The crude
peptide was cleaved from the resin according to GP 6 to give 140 mg of crude Bn-protected peptide.
After Bn-deprotection (GP 8), a portion of the crude peptide (37 mg) was purified by prep. RP-HPLC
(5 – 35% A in 5 min, 35 – 95% A in 30 min, C18) according to GP 7 to give 6 (12.2 mg) as a TFA salt.
1
Colorless solid. Anal. HPLC (5% A in 5 min, 5 – 95% A in 30 min, tR 25.75). Purity > 95%. H- and
13C-NMR spectra: see Tables 7 and 8. MALDI-MS: 1372.7 (17), 1371.7 (23, [M þ K]þ), 1356.8 (22),
1355.8 (26, [M þ Na]þ), 1335.8 (32), 1334.8 (77), 1333.8 (100, [M þ H]þ). HR-MS: 1333.7639
(C63H105N12Oþ19 ; calc. 1333.7619).
Determination of Solution Structures by NMR. All NMR measurements (including DQF-COSY,
TOCSY, HSQC, HMBC, and ROESY at tm ¼ 150, 300 ms) were conducted at 600 MHz (1H) and
150 MHz (13C) in CD3OH at 25.68 with solvent suppression by presaturation. NMR Measurements, signal
assignment, derivation of distance constraints, and simulated annealing molecular-dynamics calculations
were carried out as described previously (see, e.g., [4b]).
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Received September 10, 2009